Up to the present, as a chemical process for preparing 12-keto-cholic acid from cholic acid, a process comprising selective oxidation of a hydroxyl group of 12-position of cholic acid is known. And as a microbiological process for preparing 12-keto-cholic acid from cholic acid, a process by the use of Micrococcus (Japanese Patent Publication No. Hei 1-51998), a process by the use of Arthrobacter (Japanese Patent Publication No. Hei 1-20873, Japanese Patent Publication No. Hei 2-62234), for example, are known.
But the former chemical process for preparing 12-keto-cholic acid has problems concerning reactivity, selectivity of the reaction and safety in operations, and the process is not satisfactory with regard to its yield and the purity of products. The latter process using microorganisms is not necessarily satisfactory because microorganisms essentially assimilate cholic acid substrate and produce some by-products, and with regards to the yield and the property of the products.
As the results of our researches of microorganisms having 12-keto-cholic acid productivity and of processes for preparing 12-keto-cholic acid by the use of said microorganisms, we have eventually found that a microorganism belonging to a genus of Bacillus which may grow in a high alkaline culture medium containing high concentration of cholic acid wherein usual microorganisms may not grow produces 12-keto-cholic acid, 3.alpha., 12.alpha.-dihydroxy-7-keto-5.beta.-cholanic acid and 3.alpha.-hydroxy-7,12-diketo-5.beta.-cholanic acid as main converting products from cholic acid substrate, and we named the microorganism Bacillus sp. TTUR 2-2 (Fermentation Research Institute, Agency of Industrial Science and Technology (Japan) deposit No. 11861 (referred hereinafter as FERM1861)). This microorganism was isolated from the soil of Yonezawa city Yamagata prefecture.
Above bacterium has the ability to oxidize hydroxy groups of mainly 7-position and 12-position of cholic acid to keto groups and a hydroxy group of 3-position, though weak it is, of cholic acid to keto group, but has no ability to assimilate or decompose cholic acid.
Further as said bacterium can grow in the high alkaline medium, it is possible to prepare culture media containing high concentration of cholic acid, and Sterilization operation to the culture media prior to cultivation of bacteria, which operation is necessary for usual culture media, may be avoided.
We have attained the present invention through isolation of mutant strain having productivity of 12-ketocholic acid from cholic acid with substantially 100% conversion and substantially 100% yield by means of mutation treatment such as irradiation of ultraviolet ray, X-ray, gamma-ray and so forth, or contact with mutagenesis agent such as N-methyl-N'-nitro-N-nitrosoguanidine (referred herein after as NTG), 4-nitroquinoline-N-oxide, acriflavin, ethyl methane sulfonate and so forth.
Therefore the present invention provides a process for preparing 12-keto-cholic acid comprising: cultivating a bacterium belonging to Bacillus having 12-keto-cholic acid productivity in a nutrition medium containing cholic acid, making the bacterium produce 12-keto-cholic acid in the culture medium and collecting 12-keto-cholic acid.